Methanocarba cycloalkyl nucleoside analogues

ABSTRACT

The present invention provides novel nucleoside and nucleotide derivatives that are useful agonist or antagonists of P1 and P2 receptors. For example, the present invention provides a compound of formula A-M, wherein A is modified adenine or uracil and M is a constrained cycloalkyl group. The adenine or uracil is bonded to the constrained cycloalkyl group. The compounds of the present invention are useful in the treatment or prevention of various diseases including airway diseases (through A 2B , A 3 , P2Y 2  receptors), cancer (through A 3 , P2 receptors), cardiac arrhythmias (through A1 receptors), cardiac ischemia (through A 1 , A 3  receptors), epilepsy (through A1, P2X receptors), and Huntington&#39;s Disease (through A 2A  receptors).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.10/169,975, which is the U.S. national stage of PCT/US01/00981, filedJan. 12, 2001, claiming the benefit of U.S. Provisional Patentapplication No. 60/176,373, filed Jan. 14, 2000, the disclosures ofwhich are incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

This invention pertains to a novel class of receptor ligands for P1 andP2 receptors and their therapeutic use. More specifically, the inventionpertains to nucleoside derivatives in which the sugar moiety is replacedwith a cycloalkyl group that is conformationally constrained by fusionto a second cycloalkyl group.

BACKGROUND OF THE INVENTION

Purines such as adenosine have been shown to play a wide array of rolesin biological systems. For example, physiological roles played byadenosine include, inter alia, modulator of vasodilation andhypotension, muscle relaxant, central depressant, inhibitor of plateletaggregation, regulator of energy supply/demand, responder to oxygenavailability, neurotransmitter, and neuromodulator. (Bruns, Nucleosides& Nucleotides, 10(5), 931-934 (1991)). Because of its potent actions onmany organs and systems, adenosine and its receptors have been thesubject of considerable drug-development research (Daly, J. Med. Chem.,25, 197 (1982)). Potential therapeutic applications for agonistsinclude, for instance, the prevention of reperfusion injury aftercardiac ischemia or stroke, and treatment of hypertension and epilepsy(Jacobson, et al., J. Med. Chem., 35, 407-422 (1992)). Adenosine itselfhas recently been approved for the treatment of paroxysmal supraventricular tachycardia (Pantely, et al., Circulation, 82, 1854 (1990)).Adenosine receptor agonists also find use as anti-arrhythmics,antinociceptives, anti-lipolytics, cerebroprotectives, andantipsychotics.

P2 receptors, are present in heart, skeletal, various smooth muscles,prostate, ovary, and brain and have been implicated in certainaggregation processes associated with thrombosis and asanti-hypertensive and anti-diabetic agents. Agonists that bind the P2receptor induce activation of phospholipase C, which leads to thegeneration of inositol phosphates and diacyl glycerol with a subsequentrise in intracellular calcium concentration and muscle relaxation. P2receptor antagonists block ADP-promoted aggregation in platelets andthereby exert an anti-thrombotic effect.

All P1 and P2 receptor nucleoside ligands suffer from chemicalinstability that is caused by the labile glycosidic linkage in the sugarmoiety of the nucleoside. However, it has been found that relatively fewribose modifications are tolerated by the presently known agonists andantagonists of P1 and P2 receptors.

New compositions are needed that have improved chemical stability andthat do not destroy the activity of such compounds.

The invention provides such compositions and methods of using them inthe treatment of disease. These and other advantages of the presentinvention, as well as additional inventive features, will be apparentfrom the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel nucleoside and nucleotidederivatives that are useful agonists or antagonists of P1 or P2receptors. The invention is premised upon the novel combination ofadenine and uracil and their derivatives with a constrained cycloalkylgroup, typically a cyclopentyl group. The constraint on the cycloalkylgroup is introduced by fusion to a second cycloalkyl group. In the caseof cyclopentane, the fusion is typically with cyclopropane. The presentcompounds retain a surprising binding affinity despite the substitutionfor the ribose group. Moreover, the absence of the glycosidic bond inthe compounds assists in improving the chemical stability of thecompounds and aids in overcoming the stabilit problem associated withthe glycosidic bond in previously known P1 and P2 receptor ligands.

The compounds of the present invention are useful in the treatment orprevention of various airway diseases (through A_(2B), A₃, P2Y₂receptors), cancer (through A₃, P2 receptors), cardiac arrhythmias(through A₁ receptors), cardiac ischemia (through A₁, A₃ receptors),epilepsy (through A₁, P2X receptors), Huntington's Disease (throughA_(2A) receptors), Immunodeficient disorders (through A₂, A₃ receptors),inflammatory disorders (through A₃, P₂ receptors), neonatal hypoxia(through A₁ receptors), neurodegenerative (through A₁, A₃, P2receptors), pain (through A₁, A₃, P2X3 receptors), Parkinson's Disease(through A_(2A) receptors), renal failure (through A₁ receptors),schizophrenia (through A_(2A) receptors), sleep disorders (through A₁receptors), stroke (through A₁, A₃, P2 receptors), thrombosis (throughP2Y₁, P2Y_(AC) receptors), urinary incontinence (through P2X,receptors), diabetes (through A₁ receptors), psoriasis (through P2Xreceptors), septic shock (through P2 receptors), brain trauma (throughA₁ receptors), glaucoma (through A₃ receptors) and congestive heartfailure (through P2 receptors).

The invention may best be understood with reference to the accompanyingdrawings and in the following detailed description of the preferredembodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new class of nucleoside and nucleotideanalogs that serve as selective agonists or antagonists for P1 and P2receptors.

Generally, the compounds of the present invention comprise two basicchemical components designated “A” and “M” which are covalently bondedto one another. Component A comprises adenine or uracil, and component Mincludes a constrained cycloalkyl group. Preferably the adenine anduracil are chemically modified or substituted with moieties that allowthe compound to bind to a P1 or P2 receptor. To that end any of a widevariety of chemical groups can be used to modify adenine and uracil.Those groups are well known to those of skill in the receptor art.Preferably, when A is purine or a purine derivative, the linkage betweenA and M is a chemical bond between the N9 purine nitrogen and the C1carbon of the cycloalkyl group. Where A is pyrimidine or a pyrimidinederivative, the bond is between N1 pyrimidine nitrogen and the C1 carbonof the cycloalkyl group. The compounds of the present invention haveimproved stability and surprising receptor binding affinity.

While not wishing to be bound to any particular theory, it is believedthat the constrained cycloalkyl group assists in improving chemicalstability and receptor affinity. Preferably the cycloalkyl groups arecapable of adopting a conformation such that the compound can bind to P1or P2 receptors. As a result, preferred cycloalkyl groups are those thattend to form energetically favorable interactions with P1 and P2receptors and avoid energetically unfavorable ones, such as unfavorableionic and/or steric interactions. Further, the cycloalkyl group isderivatized with a bridging group. The constraint restricts thecycloalkyl group to certain conformations that are believed to bebeneficial to binding affinity. The preferred cycloalkyl group is acyclopentyl group. With cyclopentyl groups the preferred method forintroducing a conformational constraint is by derivatizing with a fusedcyclopropane bridge. With this modification the cyclopentane ring isbelieved to be constrained to mimic the conformation of a rigid furanosering.

Compounds of the present invention include the compounds shown below inFormulae I and II.

Formulae I and II show compounds in which a derivatized or underivatizedadenine base is joined to a constrained cyclopentyl group. For purposesof reference, the carbon atom of the cyclopentyl group, M, that isjoined to adenine, A, is the C1 carbon and the adenine is joined to Mthrough its N9 nitrogen. In the compounds of Formulae I and II theconstrained cyclopentyl group is derivatized with a fused cyclopropanebridge. In Formula I the cyclopropyl group bridges carbon atoms C4 andC6. In Formula II the cyclopropyl group bridges carbon atoms C6 and C1.These distinct bridging patterns constrain the cyclopentyl group intodistinct conformations, specifically the N-(northern) conformation as inFormula I and the S-(southern) conformation as in Formula II. These twoconformations are thought to mimic the two biologically activeconformations of furanose groups for P1 and P2 receptor binding pockets.

The compounds described by Formulae I and II can be further defined by avariety of suitable modifications to the adenine group. As discussedabove, any of a wide variety of chemical groups can be used to formsuitable adenine derivatives that comprise the novel compounds of thepresent invention, provided that the resulting compound is capable ofbinding to a P1 or P2 receptor. These chemical groups are well known inthe art and have been described, for example in U.S. Pat. Nos.5,284,834; 5,498,605; 5,620,676; 5,688,774; and Jacobson and Van Rhee,PURINERGIC APPROACHES IN EXPERIMENTAL THERAPEUTICS, Chapter 6, p. 101(Jacobson and Jarvis eds., 1997); and Jacobson et al., THE P2 NUCLEOTIDERECEPTORS, p. 81-107, in THE RECEPTORS (Turner et al. eds. 1998), whichare incorporated by reference herein. The combination of the chemicallymodified adenine and the constrained cycloalkyl group provides asurprising improvement in both chemical stability and binding affinity.

By way of example and not in limitation of the present invention in thecompounds of Formulae I and II, R₁ is hydrogen, alkyl, cycloalkyl,alkoxy, cycloalkoxy, aryl, arylalkyl, acyl, sulfonyl, arylsulfonyl,thiazolyl or bicyclic alkyl; R₂ is hydrogen, halo, alkyl, aryl,arylamino, aryloxide, alkynyl, alkenyl, thioether, cyano, alkylthio orarylalkylthio; R₃, R₄, and R₅, are each hydrogen, hydroxyl, alkoxy,alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino, arylamino, phosphoryl,phosphonyl, boronyl, or vanadyl, and they can be the same or different;R₆ is hydrogen, alkyl, alkenyl, alkynyl, or aminoalkyl. R₇ is amethylene, dihalomethyl, carbonyl, or sulfoxide group. R₈ is carbon ornitrogen. At least one of R₁, R₂, and R₆ is not hydrogen. It can beappreciated that various combinations of the above groups are alsowithin the invention provided that they retain agonist or antagonistactivity with a P1 or P2 type receptor.

Where an alkyl, alkenyl, alkynyl group is referenced by itself or aspart of another group, the reference is to an uninterrupted carbon chainconsisting of no more than 20 carbon atoms. Aryl and cycloalkyl groupscontain no more than 8 carbons in the ring.

Reference to alkyl groups is further meant to include straight orbranched chain alkyls, arylalkyl, aminoalkyl, haloalkyl, alkylthio orarylalkylthio groups. Alkyls specifically include methyl throughdodecyl. Where alkyl groups are present at position R₆ in adenine, it ispreferred that the chain length be no longer than 6 carbons. Arylalkylgroups include, phenylisopropyl, phenylethyl. Aminoalkyl groups can beany suitable alkyl group also containing an amine. Similarly, haloalkylgroups can be any suitable alkyl group that contains a halo substituent,such as bromo, chloro, flouro, iodo. Alkylthio includes such moieties asthiomethyl, thiopentyl, thiohexyl, thioheptyl, thiooctyl, thiodecyl,thioundecyl, ethylthioethyl, or 6-cyanohexylthio groups. Alkylthio alsois meant to include arylalkylthio such as 2-(p-nitrophenyl)ethyl)thio,2-aminophenylethylthio, 2-(p-nitrophenyl)ethylthio, or2-aminophenylethylthio.

Cycloalkyls for example cyclopentyl, cyclohexyl, hydroxycyclopentyl.

Alkoxys include for example methoxy groups.

Cycloalkoxys can include cyclopentoxy.

Aryl moieties can be arylalkyl, arylalkylthio, arylsulfonyl, arylamino,aryloxide, heteroaryl, haloaryl, arylurea, arylcarboxamido,heteroarylamino or sulfoaryl. Benzyl groups are one species of arylgroup. In addition, the arylalkyls include R-phenylisopropyl orphenylethyl. Aryloxides can be phenyl, R-phenylisopropyl, phenylethyl,3,5-dimethoxyphenyl-2-(2-methylphenyl)ethyl and sulfophenyl. Haloarylcan be iodobenzyl among other halogenated aryl groups. Additionally, theheteroaryls include, for example, furans such as tetrahydrofuran.

Acyl groups include carbonyls.

Alkenyl groups are analogous to alkyl groups but include at least onecarbon-carbon double bond. When present at the R₆ group of adenine it ispreferred that the carbon chain length be from 2 to 6 carbons.

Similarly, alkynyls are analogous to alkenyl groups but contain at leastone triple carbon-carbon bond. As with other groups, when present at theR₆ position of adenine it is preferred that they are not longer than 6carbons.

Phosphoryl groups include diphosphoryl, triphosphoryl, thiophosphoryl,thiodiphosphoryl, thiotriphosphoryl, imidodiphosphate,imidotriphosphate, methylene diphosphate, methylenetriphosphate,halomethylene diphosphate, halomethylene triphosphate, boranophosphate,boranodiphosphate, boranotriphosphate, or phosphorothioate-2-thioetherfor example.

Thio groups include alkylthio, arylalkylthio, alkenylthio, or arylthios.Alkylthio includes such groups as thiomethyl, thiopentyl, thiohexyl,thioheptyl, thiooctyl, thiodecyl, thioundecyl, ethylthioethyl, or6-cyanohexylthio. Alkenylthio includes 5-hexenylthio. Arylthios include2-(p-nitrophenyl)ethyl)thio, 2-aminophenylethylthio,2-(p-nitrophenyl)ethylthio, or 2-aminophenylethylthio.

One example of a suitable thiazolyl is (benzothiazolyl)thio-2-propyl.

Examples of bicycloalkyls include s-endonorbornyl, orcarbamethylcyclopentane.

Halo groups include such elements as fluoro, bromo, chloro, or iodo.

It will also be appreciated that any group that may be furthersubstituted can be, and still be within the scope of the invention. Forexample, all of the R₁ groups except hydrogen can be furthersubstituted. By way of illustration, when R₁ is not hydrogen, it can befurther modified by substitutions with any of the following chemicalsubstituents including amino, cyano, alkoxyl, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, arylalkyl, acyl, halo, hydroxy, phosphoryl, sulfonyl,sulfonamido, carboxyl, thiohydroxyl, sulfonamido, carboxyl, andcarboxamido groups. Similarly, for R₂-R₁₀ all of the groups other thanhydrogen can be substituted further. Multiple substitutions are alsocontemplated.

In a preferred embodiment R₁ can be either methyl, cyclopentyl,cyclohexyl, phenyl, R-phenylisopropyl, benzyl, or phenylethyl; R₂ ischloride; and R₆ can be a C₁-C₆ alkylamino, C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl group.

Other compounds of the present invention include the compounds shownbelow in Formulae III and IV. The Formulae show compounds in which aderivatized or underivatized uracil base is joined to a constrainedcyclopentyl group.

The compounds defined by formulae III and IV can be further defined by avariety of suitable modifications. For example R₁ can be hydrogen, or analkyl group; R₂ can be hydrogen, C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆alkynyl, or a C₁-C₆ aminoalkyl group; R₃, R₄, R₅, can each independentlybe the same as discussed previously with respect to Formulae 1 andFormulae II. R₆ and R₇ are each independently either sulfur or oxygen.

Certain compounds of the present invention are ligands of P2 receptors.A variety of P2 receptors are known in the art and the present compoundsact at one or more of these, which include for example, P2X and P2Yreceptors. These Receptor ligands are compounds that bind receptors,preferably in the binding pocket. In certain embodiments the compoundcan be a P2 receptor agonist. In other embodiments the compound can be aP2 receptor antagonist.

Certain compounds of the present invention are ligands for the P1receptor. A variety of subclasses of P1 receptors are known and variousof present compounds act at one or more these species, which include forexample A₁, A₂, and A₃ receptors. Certain compounds act as P1 receptoragonists while others appear to act as antagonists.

The compounds of the present invention are useful in the treatment orprevention of various airway diseases (through A_(2B), A₃, P2Y₂receptors), cancer (through A₃, P2 receptors), cardiac arrhythmias(through A₁ receptors), cardiac ischemia (through A₁, A₃ receptors),epilepsy (through A₁, P2X receptors), Huntington's Disease (throughA_(2A) receptors), Immunodeficient disorders (through A₂, A₃ receptors),inflammatory disorders (through A₃, P₂ receptors), neonatal hypoxia(through A₁ receptors), neurodegenerative (through A₁, A₃, P2receptors), pain (through A₁, A₃, P2X3 receptors), Parkinson's Disease(through A_(2A) receptors), renal failure (through A₁ receptors),schizophrenia (through A_(2A) receptors), sleep disorders (through A₁receptors), stroke (through A₁, A₃, P2 receptors), thrombosis (throughP2Y₁, P²Y_(AC) receptors), urinary incontinence (through P2X,receptors), diabetes (through A₁ receptors), psoriasis (through P2Xreceptors), septic shock (through P2 receptors), brain trauma (throughA₁ receptors), glaucoma (through A₃ receptors), and congestive heartfailure (through P2 receptors).

The present invention is further directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one compound selected from the group consisting of the presentlydescribed compounds.

The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone that is chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use.

The choice of excipient will be determined in part by the particularcompound of the present invention chosen, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the pharmaceutical composition ofthe present invention. The following formulations for oral, aerosol,parenteral, subcutaneous, intravenous, intramuscular, interperitoneal,rectal, and vaginal administration are merely exemplary and are in noway limiting.

One skilled in the art will appreciate that suitable methods ofutilizing a compound and administering it to a mammal for the treatmentof disease states, which would be useful in the method of the presentinvention, are available. Although more than one route can be used toadminister a particular compound, a particular route can provide a moreimmediate and more effective reaction than another route. Accordingly,the described methods are merely exemplary and are in no way limiting.

The dose administered to an animal, particularly human and othermammals, in accordance with the present invention should be sufficientto effect the desired response. Such responses include reversal orprevention of the bad effects of the disease for which treatment isdesired or to elicit the desired benefit One skilled in the art willrecognize that dosage will depend upon a variety of factors, includingthe age, species, condition or disease state, and body weight of theanimal, as well as the source and extent of the disease condition in theanimal. The size of the dose will also be determined by the route,timing and frequency of administration as well as the existence, nature,and extent of any adverse side-effects that might accompany theadministration of a particular compound and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousconditions or disease states may require prolonged treatment involvingmultiple administrations.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 to about 300 mg of one or moreof the compounds described above per kg body weight of the individual.

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope. In theexamples, unless otherwise noted, compounds were characterized andresonances assigned by 300 MHz proton nuclear magnetic resonance massspectroscopy using a Varian GEMINI-300 FT-NMR spectrometer. Also, unlessnoted otherwise, chemical shifts are expressed as ppm downfield fromtetramethylsilane. Synthetic intermediates were characterized bychemical ionization mass spectrometry (NH₃) and adenosine derivatives byfast atom bombardment mass spectrometry (positive ions in a noba orm-bullet matrix) on a JEOL SX102 mass spectrometer. Low resolutionCI—NH₃ (chemical ionization) mass spectra were carried out with Finnigan4600 mass spectrometer and high-resolution EI (electron impact) massspectrometry with a VG7070F mass spectrometry at 6 kV. Elementalanalysis was performed by Atlantic Microlab Inc. (Norcross, Ga.). NMRand mass spectra were consistent with the assigned structure.

EXAMPLE 1

In all of the potent adenosine agonists previously developed, the ribosemoiety is present, and consequently, these agonists are subject todeglycosylation and other pathways of metabolic degradation in vivo. Inorder to design non-glycosyl adenosine agonists and thereby increasebiological stability and potential receptor selectivity, carbocyclicmodifications of the ribose moiety have been introduced. In previousstudies of adenosine analogues it was found that if adenosinederivatives having carbocyclic modifications of the ribose ring(compounds 1-4, below) bind to adenosine receptors it is only withgreatly reduced affinity.

In the present study we have incorporated a complex carbocyclicmodification of ribose for use with adenosine agonists. Thismodification, wherein only one isomeric form retains high affinity andreceptor selectivity, is the “methanocarba” ring. In this modification afused cyclopropane ring constrains the accompanying cyclopentane moietyto mimic the conformation of a rigid furanose ring. The furanose ring ofnucleosides and nucleotides in solution is known to exist in a rapid,dynamic equilibrium between a range of Northern and opposing Southernconformations as defined in the pseudorotational cycle. For methanocarbaanalogues, the bicyclo[3.1.0]hexane ring can constrain the cyclopentanering into a N-, 2′-exo envelope pucker, and a S-, 3′ exo form.

These two extreme forms of ring pucker usually define biologicallyactive conformations. This example shows that nucleoside binding toP1-(adenosine) receptors, is favored when the fixed ring-twistconformation is in the N-conformation.

Chemical Synthesis.

Nucleosides and synthetic reagents were purchased from Sigma ChemicalCo. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.). 2,6-Dichloropurinewas obtained from Sigma. m-iodobenzyl bromide was purchased from Aldrich(St. Louis, Mo.).4-(6-Aminopurin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol (1)and compounds 5c and 5d were obtained from Dr. Victor Marquez. Compounds7a and 9a were synthesized in our laboratory.

The synthetic strategy used in this example is shown below. Thesynthesis of N6-substituted N-methanocarba adenosine derivativesoptimized for interaction with A1 (CP=cyclopentyl) or A3(IB=3-iodobenzyl) receptors. Reagents: a) DEAD, Ph₃P; b) MEOH, rt; c)BCl₃; d) H2/Pd; e) 3-iodobenzyl bromide, 50° C., DMF, 2 days; f) NH₄0H,MEOH, 80° C., 3 days.

(1′R,2R,3′R,4′R,1′aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(6c)

A solution of 8c (4 mg, 0.01 mmol) in methanol (0.5 ml) was hydrogenatedat atmospheric pressure over 10% Pd/C (1 mg) to furnish the product 6c(83% yield). H′NMR (CD₃0D): δ 0.7-0.8 (m, IH, 6′-CHH), 1.46-1.88 (m,1OH, 6′CHH, 1′aH, 4CH₂),2.01-2.20 (m, 1H, NCH), 3.34 (d, 1H, J=9.77 Hz,5′CHH), 3.88 (d, 1H, J=6.84 Hz, 3′CH), 4.26 (d, 1H, J=9.77 Hz, 5′CHH),4.66-498 (m, 2H, 2′CH, 1′CH), 8.28 (s, 1H, 2CH), 8.5 (s, 1H, 8CH).HRMS(FAB): Cal: 346.1879 Found: 346.1879

(1′R,2′R,3′R,4′R)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(6-(3-idobenzylamino)purine-9-yl)cyclopentane(7b)

A mixture of aristeromycin (3.5 mg, 0.013 mmol) and 3-iodobenzybromide(12 mg, 0.039 mmol) in anhydrous DMF was heated for 3 days, and solventwas removed under vacuum. The excess 3-iodobenzylamine was removed fromthe reaction mixture by adding ether to the reaction mixture, andstirring was continued for 5 min. followed by decantation of thesupernatant ether phase. The residue was dried, suspended in methanol (1ml) and ammonium hydroxide (0.5 ml), and heated at 80° C. in a closedtube for 1 h. Solvent was removed under vacuum, and the residue obtainedwas purified by flash column chromatography using 7/3chloroform/methanol to furnish 3.0 mg (47%) of the product.

H¹NMR(CD₃0D) δ 1.86-1.96 (m, 1H, 1′CHH), 2.14-2.30 (m, 1H, 1′CHH),2.38-2.48 (m, 1H, 4′CH), 3.3-3.38 (m, 1H, 5′CHH), 3.67 (d, 1H, J=6.84Hz, 5′CHH), 3.96-4.06 (m, 1H, 3′CH), 4.43-4.48 (m, 1H, 2′CH), 4.73-4.82(m, 1H, 1′CH), 5.26 (s, 2H, ArCH₂), 7.12 (t, 1H, J=7.82 Hz, ArH), 7.32(d, 1H, J=7.82 Hz, ArH), 7.66 (d, 1H, J=7.82 Hz, ArH), 7.73 (s, 1H,ArH), 8.06 (s, 1H, 2CH). 8.08 (s, 1H, 8CH).

Preparation of4-[6-(3-iodobenzylamino)-purin-9-yl]-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol(7c, (N)-Methanocarba-N⁶-(3-iodobenzyl)adenosine) by Dimrothrarrangement:¹

To a solution of4-(6-amino-purin-9-yl)-1-hydroxymethyl-bicyclo[3.1.0]hexane-2,3-diol(5c, 20 mg, 0.0721 mmol) in DMF (0.5 mL) was added m-iodobenzyl bromide(64 mg, 0.216 mmol), and the mixture was stirred at 50° C. for 2 days.DMF was then removed under a stream of N₂. To the resulting syrup 0.5 mLof acetone and 1 mL of ether were added and the syrup solidified. Thesolvents were removed by decantation, and again ether was added andremoved. The solid was dried and dissolved in 1 mL MEOH. NH₄OH (1.5 mL)was added and the mixture was stirred at 80° C. for 3 days. Aftercooling down to room temperature, the solvents were removed underreduced pressure and the residue was purified by preparative TLC (silica60; 1 000 μm; Analtech, Newark, Del.; ethyl acetate-i-PrOH—H₂O (8:2:1))to give 26 mg of the product (7c), yield: 73%. ¹H NMR (CDC1₃): δ 0.82(t, J=6.0 Hz, 1H), 1.41 (t, J=4.8 Hz, 1H), 1.72 (dd, J=8.5, 6.0 Hz, 1H),3.36 (d, J=10.8 Hz, 1H), 4.05 (d, J=6.9 Hz, 1H), 4.33 (m, 1H), 4.80-4.88(m, 3H), 5.21 (d, J=6.9 Hz, 1H), 6.25 (m, br, 1), 7.07 (t, J=7.8 Hz,1H), 7.35 (d, J=7.8 Hz, 1H), 7.61 (d, J=7.8 Hz, 1H), 7.74 (s, 1), 7.93(s, 1H), 8.33 (s, 1H). MS(FAB): m/z 494 (M⁺+I).

(1′R,2′R,3′R,4′R,1′aR,)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(8c):

To a solution of 15 (36 mg, 0.076 mmol) in anhydrous dichloromethane wasadded BCl₃ (1M solution in dichloromethane, 0.23 ml, 0.23 mmol) at 0° C.The reaction mixture was warmed to room temperature and stirred for 10min. To this mixture was added methanol (1 ml) followed by ammoniumhydroxide (0.5 ml). The mixture was concentrated under vacuum, and theresidue obtained was purified by flash column chromatography using 9/1chloroform-1/methanol as eluent to furnish 14 mg of the product 8c (48%yield) as a solid.

H¹NMR(CDCl₃): δ.o.65-0.9 (m, 1H, 6′CHH), 1.1-1.4 (m, 2H, 6′CHH, 1′aH),1.4-1.9 (m, 8H, 4CH₂), 2.0-2.2 (m, 1H, N⁶CH), 3.34 (d, 1H, J=7.2 Hz,5′CHH), 3.97 (d, 1H, J=4.6 Hz, 3′CH), 4.25 (d, 1H, J=7.2 Hz,5′CHH),4.687 (s, 1H, 1′CH), 5.11 (d, 1H, J 4.6, 2′CH), 7.85 (s, 1H,8CH). HRMS(FAB): Cal: 380.1489 found: 380.1498

(1′R,2′R,3′R,4′R,1′aR)-2,3-(dihydroxy)-4-(hydroxymethyl)-1-(2-chloro-6-(3-idobenzylamino)purine-9-yl)bicyclo(3.1.0)hexane)(9c) was synthesized by the same method as 8c in 53% yield.

H¹NMR(CD₃OD): δ 0.70-0.78 (m, 1H, 6′CHH), 1.50-1.63 (m, 2H, 6, CHH,1′aH), 3.33 (d, 1H, J=11.72 Hz, 5′CHH), 3.88 (d, 1H, J=6.84 Hz, 3′CH),4.26 (d, 1H, J=11.72 Hz, 5′CHH), 4.71-4.83 (m, 2H, 1′CH, 2′CH), 7.1 (t,1H, J=7.82 Hz, ArH), 7.40 (d, 1H, J=7.82 Hz, ArH), 7.61 (d, 1H, 7.82 Hz,ArH), 7.78 (s, 1H, ArH), 8.54 (s, 1H, 8CH). HRMS(FAB): Cal: 528.0299Found: 528.0295

(2R,3R,4R,1′aR,1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2,6dichloropurine-9-yl)bicyclo(3.1.0)hexane)(12)

To a solution of triphenyl phosphine (260 mg, 1 mmol) in anhydrous THF(2 ml) was added DEAD (0.16 ml, 1 mmol) dropwise at 0° C., and stirringwas continued for 20 min. To this solution was added a solution of2,6-dichloropurine in THF (4 ml) followed by the addition of 11 (145 mg,0.5 mmol) in THF (4 ml). The reaction mixture was warmed to roomtemperature, and stirring was continued for 6 h. Solvent was evaporatedunder vacuum, and the residue obtained was purified by flashchromatography using 7/3 petroleumether/ethylacetate as eluent tofurnish 141 mg of the product (12) (70% yield) as a gum.

H¹NMR (CDCl₃): ∂ 1.0 (m,1H, 6′CHH), 1.24 (s, 3H,CH₃), 1.27-1.38 (m, 1H,6′CHH), 1.55 (s, 3H, CH₃),1.62 (dd, 1H, J=4.88, 9.77 Hz, 1′aH), 3.34 (d,1H, J=9.77 Hz, 5′CHH), 3.97 (d, 1H, J=9.77 Hz, 5′CHH), 4.50 (d, 1H,J=6.84 Hz, 3′CH), 4.57-4.68 (qAB, 2H, J=12.7 Hz, ArCH₂), 5.17 (s, 1H,1′CH), 5.32 (d, 1H, J=6.84 Hz, 2′H), 7.27.4 (m, 5H, Ar), 8.63 (s, 1H,8CH).

(2R,3R,4R,1′aR,1S)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-cyclopentylaminopurine-9-yl)bicyclo(3.1.0)hexane)(15):

To a solution of 12 (42 mg, 0.105 mmol) in methanol (2 ml) was addedcyclopentylamine at room temperature, and stirring was continued for 6hr for complete reaction. Solvent was removed under vacuum, and theresidue obtained was purified by flash column chromatography using 7/3petroleum ether/ethylacetate as eluent to furnish 45 mg of the product15 (90% yield) as a gum.

H¹NMR(CDCl₃): δ 0.92-0.96 (m, 1H. 6′CHH), 1.14-1.01 (m, 1H, 6′CHH), 1.23(s, 3H, CH₃),1.42-1.81 (m, 9H, 1′aH, 4CH₂),1.54 (s, 3H, CH₃), 2.08-2.21(m, 1H, N⁶CH), 3.44 (d, 1H, J=9.76 Hz, 5′CHH), 3.90 (d, 1H, J=9.76 Hz,5′CHH), 4.51 (d, 1H, J=6.84 Hz, 3′CH), 4.57-4.67 (qAB, 2H, J=12.7 Hz,ArCH₂), 5.04 (s, 1H, 1′CH), 5.32 (d, 1H, J=6.84 Hz, 2′CH), 7.2-7.4 (m,5H, Ar), 8.18 (s, 1H, 8CH).

(1′R,2′R,3′R,4′R,1′aR)-2,3-(O-isopropylidine)-4-(methylenebenzyloxy)-1-(2-chloro-6-(3-idobenzylamino)purine-9-yl)bicyclo(3.1.0)hexane)(16) was synthesized in 70% yield by the same method as 15, except using3-iodobenzylamine hydrochloride and two equivalents of triethylamine.

H¹NMR(CDCl₃): δ 0.87-0.91 (m, 1H, 6′CHH), 1.10-1.29 (m, 1H, 6′CHH), 1.17(s, 3H, CH₃),1.42-1.56 (m, 1H, 1′aH), 1.47 (s, 3H, CH₃), 3.37 (d, 1H,J=9.77 Hz, 5′CHH), 3.84 (d, 1H, J=9.77 Hz, 5′CHH), 4.44 (d, 1H, J=6.84Hz, 3′CH), 4.50-4.60 (qAB, 2H, J=11.72 Hz, ArCH₂), 4.70 (bs, 1H, NH),4.98 (s, 1H, 1′CH), 5.24 (d, 1H, J=6.84 Hz, 2′CH), 7.0 (t, 1H, J=7.82Hz, ArH), 7.2-7.34 (m, 6H, ArH), 7.55 (d, 1H, J=7.82, ArH), 7.65 (s, 1H,ArH), 8.08 (s, 1H, 8CH).

Pharmacological Analyses.

Materials

F-12 (Ham's) medium, fetal bovine serum (FBS) andpenicillin/streptomycin were from Gibco BRL (Gaithersburg, Md.). [¹²⁵I]AB-MECA (1000 Ci/mmol) and [³⁵S]guanosine 5′-(γ-thio)triphosphate(1000-1500 Ci/mmol) were from DuPont NEN (Boston, Mass.). Adenosinedeaminase (ADA) was from Boehringer Mannheim (Indianapolis, Ind.). Allother materials were from standard local sources and of the highestgrade commercially available.

Cell Culture and Membrane Preparation

CHO cells stably transfected with either human A₁ or A₃ receptors (giftof Dr. Gary Stiles and Dr. Mark Olah, Duke University Medical Center)were cultured as monolayers in medium supplemented with 10% fetal bovineserum. Cells were washed twice with 10 ml of ice-cold phosphate bufferedsaline, lysed in lysis buffer (10 mM Tris.HCl buffer, pH 7.4, containing2 mM MgCl₂ and 0.5 mM EDTA), and homogenized in a Polytron homogenizerin the presence of 0.2 U/ml adenosine deaminase. The crude membraneswere prepared by centrifuging the homogenate at 1000×g for 10 minfollowed by centrifugation of the supernatant at 40,000×g for 15 min.The pellet was washed once with the lysis buffer and recentrifuged at40,000×g for 15 min. The final pellets were resuspended in 50 mMTris.HCl buffer, pH 7.4, containing 10 mM MgCl₂ and 0.1 mM EDTA andstored at −70° C.

Radioreceptor Binding

Determination of binding to adenosine A₁, A_(2A) and A_(2B) receptorswas carried out as reported. Determination of A₃ adenosine receptorbinding was carried out using [¹²⁵I]AB-MECA. Briefly, aliquots of crudetransfected CHO cell membranes (approximately 40 μg protein/tube) wereincubated with 0.5 nM [¹²⁵I]AB-MECA, 10 mM MgC1₂, 2 units/ml adenosinedeaminase, 50 mM Tris.HCl (pH 7.4) at 37° C. for 60 min. The totalvolume of the reaction mixture was 125 μl. Bound and free ligands wereseparated by rapid filtration of the reaction mixture through WhatmanGF/B glass filters. The filters were immediately washed with two 5ml-portions of ice-cold 50 mM Tris.HCl buffer (pH 7.4). Theradioactivity bound to the filters was determined in a Beckman gammacounter. Specific binding was defined as the amount of the radioligandbound in the absence of competing ligand minus the amount of that boundin the presence of 100 μM NECA. Ki-values were calculated using theK_(d) for [¹²⁵I]AB-MECA binding of 0.56 nM.

Determination of [3′S]GTPγS Binding

[³⁵ S]GTPγS binding was determined by the method of Lorenzen et al. Theincubation mixture contained in a total volume of 125 μl, 50 mM Tris.HCl(pH 7.4), 1 mM EDTA, 10 mM MgCl₂, 10 μM guanosine 5′-diphosphate, 1 mMdithiothreitol, 100 mM NaCl, 0.2 units/ml adenosine deaminase, 0.16 nM[³⁵ S] GTPγS (about 50,000 cpm) and 0.5% BSA. The CHO cell membranesexpressing A₁ or A₃ receptors were preincubated with the above-mentionedassay mixture at 37° C. for 1 h and further incubated for 1 hr after theaddition of [³⁵ S]GTPγS. Incubations were terminated by rapid filtrationof the samples through glass fiber filters (Whatman GF/B), followed bytwo 5 ml washes of the same buffer. After transferring the filters intoa vial containing 3 ml of scintillation cocktail, the radioactivity wasdetermined in a scintillation counter.

Data analysis. Analyses of saturation binding assays andconcentration-response curves were carried out using the GraphPad Prism(GraphPad Software Inc., San Diego, Calif.). Comparisons between groupswere carried out using the unpaired Student's test.

Results

Chemical Synthesis

The methanocarbocyclic 2′-deoxyadenosine analogues, shown below in Table1, in which a fused cyclopropane ring constrains the cyclopentane ringinto a rigid envelope configuration of either a N- or S-conformation,were synthesized in a manner similar as shown above. The N-methanocarbaanalogues of various N⁶-substituted adenosine derivatives, includingcyclopentyl and iodobenzyl, in which the parent compounds are potent andselective agonists at either A₁ or A₃ receptors, respectively, wereprepared. 2,6-Dichloropurine, 10, was condensed with the cyclopentylderivative, 11, using the Mitsunobu reaction, followed by substitutionat the 6-position and deprotection to give 8c or 9e. The 2-chlorosubstitution of compound 8c was removed by catalytic reduction to give6c. This allowed the incorporation in the N-configuration series of the2-chloro modification of adenine, which was of interest for its effecton adenosine receptor affinity. An N⁶-(3-iodobenzyl) group could also beintroduced in either aristeromycin, 5b, or N-methanocarba-adenosine, 5c,by the Dimroth rearrangement, to give 7b and 7c.

Biological Activity

A pair of methanocarba analogues of adenosine, 5c and 5d, correspondingto N- and S-conformations of ribose, were tested in binding assays, theresults of which are shown in Table 1 below, at four subtypes ofadenosine receptors. The more synthetically challenging S-isomer (5d)was available only as the racemate and therefore was tested as such. Atrat A1, rat A2A, and human A3 subtypes, the N-analogue proved to be ofmuch higher affinity than the S-analogue. At the human A2B receptor,binding was carried out using [3H]ZM 241,385, however the affinity wastoo weak to establish selectivity for a specific isomer. Affinity ofN-methanocarba-adenosine, 5c, vs. adenosine, 5a, was particularlyenhanced at the A3 receptor subtype, for which the ratio of affinitiesof N- to S-analogues was 150-fold. Although a poor substrate foradenosine deaminase (ADA), the binding curve for 5c was shifted in thepresence of ADA, therefore the affinity values for 5c and 5d obtained inthe absence of ADA are entered in Table 1, below. The South confomer,5d, is even a worse substrate of ADA (100-fold less) which explains whythe curves in the presence and absence of ADA for 5d are virtually thesame. Aristeromycin, 5b, bound weakly to adenosine receptors, withslight selectivity for the A_(2A) subtype. Compound 5c was more potentthan aristeromycin, 5b, in binding to A1 (4-fold) and A3 (4500-fold)adenosine receptors.

Compounds 6c and 8c are patterned after A1 receptor-selective agonists,while compounds 7c and 9c are patterned after A3 receptor-selectiveagonists. Compounds 6 and 7 are unsubstituted at the 2-position, whilecompounds 8 and 9 contain the potency enhancing 2-chloro substituent.The N6-cyclopentyl N-methanocarba derivative, 6c, based on CPA, 6a,maintained high selectivity for A1 receptors, although the affinity of6c at rat A1 receptors was 3-fold less than for 6a. In one series it waspossible to compare ribose, cyclopentyl, and N-methanocarba derivativeshaving the same N6-substitution. The N6-(3-iodobenzyl) derivative, 7c,based on a 5′-hydroxy analogue, 7a, of IB-MECA, with a Ki value of 4.1nM was 2.3-fold more potent at A3 receptors than the ribose-containingparent. Thus, the selectivity of 7c for human A3 versus rat AI receptorswas 17-fold. The aristeromycin analogue, 7b, was relatively weak inbinding to adenosine receptors.

Among 2-chloro-substituted derivatives, the N-methanocarba analogue, 8c,was less potent at A1 and A2A receptors than its parent2-chloro-N-6-cyclopentyladenosine, 8a, and roughly equipotent at A3receptors. Thus, 8c was 53-fold selective in binding to rat A1 vs. humanA3 receptors. The N-methanocarba analogue, 9c, of2-chloro-N-6-(3iodobenzyl)adenosine, 9a, had Ki values (nM) of 141, 732,and 2.2 at A1, A2A, and A3 receptors, respectively. Thus, the 2-chlorogroup slightly enhanced affinity at A3 receptors, while reducingaffinity at A1 receptors.

The receptor binding affinity upon replacement of ribose with theN-methanocarba moiety was best preserved for the A3 subtype, at whichdifferences were small. At A1 receptors the loss of affinity forstructures 6-9 was between 3- and 8-fold. At A2A receptors the loss ofaffinity was between 6- and 34-fold.

The agonist-induced stimulation of binding of guanine nucleotides toactivated G-proteins has been used as a functional assay for a varietyof receptors, including adenosine receptors. Binding of [³⁵S]GTP-γ-S wasstudied in membranes prepared from CHO cells stably expressing human A1or A3 receptors (Table 2). The non-selective adenosine agonist NECA(5′-N-ethyluronamidoadenosine) caused a concentration-dependent increasein the level of the guanine nucleotide bound. Compound 6c was highlyselective and a full agonist at human A1 but not rat A1 receptors. Both7c and 9c stimulated the binding of [³⁵S]GTP-γ-S, however the maximalstimulation was significantly less than that produced by either NECA orN6(3-iodobenzyl)adenosine, 7a, both being full A3 agonists. Compounds 7cand 9c resulted in relative stimulation of [³⁵S]GTP-γ-S binding of only45% and 22%, respectively, indicating that the efficacy of theN-methanocarba analogue at A3 receptors was further reduced upon2-chloro modification. The potency of compounds 7c and 9c, indicated bythe EC50 values in this functional assay, was greater than the potenciesof either NECA or compound 7a (Table 2). Thus, the N-methanocarbaN6-(3-iodobenzyl) analogues appear to be highly potent and selectivepartial agonists at human A3 receptors TABLE I Affinities of AdenosineDerivatives¹

K₁ (nM) or % displacement Compound R′ R RA₁ ^(a) RA_(2A) ^(b) hA₃ ^(c)1a H cyclopentyl 0.59 462 274 ± 20    CPA 240 (r) 1B H cyclopentyl 5.06± 0.51 6800 ± 1800 170 ± 51    1781 1c H cyclopentyl 5110 ± 790  15% at10 μM 1783 2a H 3-iodobenzyl 20.0 ± 8.5  17.5 ± 0.5  9.5 ± 1.4 (r)IBO-ADO, 541 2b H 3-iodobenzyl 69.2 ± 9.8  601 ± 236 4.13 ± 1.76   17433a Cl yclopentyl 0.6 950 237 (r) CCPA 3b Cl cyclopentyl 8.76 ± 0.81 3390± 520  466 ± 58    1761 3c Cl cyclopentyl 3600 ± 780  45 ± 5% at 100 μM1782 4a Cl 3-iodobenzyl 18.5 ± 4.7  38.5 ± 2.0  1.41 ± 0.17 (r) 542 4bCl 3-iodobenzyl 141 ± 22  732 ± 207 2.24 ± 1.45    1760 4c Cl3-iodobenzyl 8730 ± 370  25,400 ± 3800   1784 Compound R₂ rA₁ ^(a)rA_(2A) ^(b) hA_(2B) ^(b) hA₃ ^(b) A₁/A₃ 5a H Estd. 10^(d) estd.30^(d)<10% at 100 μM estd. 1000(r)^(d,e) 100 5b H 6260 ± 730  2150 ± 950 47,300 ± 10,600   20,000 ± 7900 (r)^(e) 0.31 5c H 1680 ± 80    22,500 ±100 (h)^(e,f) 35 ± 2% at 50 μM^(f) 404 ± 70^(f)  4.2 5d H 15% at 100μM >100,000 (h)^(e,f) 20 ± 4% at 50 μM^(f) 62,500 ± 2900^(f)   >1(racemic) 6a CP 1.50 ± 0.51 857 ± 163 21,200 ± 4300       274 ± 20,240(r)^(e) 0.0055 6c CP 5.06 ± 0.51 6800 ± 1800 139k ± 19k  170 ± 51  0.0307a IB 20.0 ± 8.5  17.5 ± 0.5  3570 ± 100    9.5 ± 1.4 (r)^(e) 2.1 7b IB25,900 ± 1600   <10% 100 μM n.d. 1960 ± 370  13 7c IB 69.2 ± 9.8  601 ±236 12,100 ± 1300   4.13 ± 1.76 17 8a CP 1.33 ± 0.19 605 ± 154 20,400 ±1200   237 (r)^(e) 0.0056 8c CP 8.76 ± 0.81 3390 ± 520  27 ± 7% at 100μM 466 ± 58  0.019 9a IB 18.5 ± 4.7  38.5 ± 2.0  5010 ± 1400   1.41 ±0.17 (r)^(e) 13 9c IB 141 ± 22  732 ± 207 41,000 ± 700   2.24 ± 1.45 63¹(a) simple carbocyclic, (b) and methanocarba-adenosine,(N)-conformation, (c) and S-conformation, (d) derivatives in radioligandbinding assays at rat A₁,^(a) rat A_(2A),^(b) human A_(2B),^(b) andhuman A₃ receptors, ^(c) unless noted.^(e)

TABLE II Effect of ligands to stimulate [³⁵S]GTPγS binding to membranesof cells expressing the cloned hA₁AR or hA₃AR or in rat cerebralcortical membranes containing the A₁AR cloned hA₁AR % Maximal rA₁AR %Maximal cloned hA₃AR % Maximal Ligand EC₅₀ (nM)^(a) Stimulation^(c) EC₅₀(nM)^(a) Stimulation^(c) EC₅₀ (nM)^(a) Stimulation^(c) NECA n.d. n.d.155 ± 15  100 6a  4.15 ± 0.90 100  20.3 ± 13.1 100 7980 ± 60  100 6c21.5 ± 2.3 102 ± 1  100 ± 17 75 ± 6 >10,000 14 ± 2% at 10 μM 7a  43.1 ±10.4 91 ± 1 340 ± 98 95 ± 4 5.16 ± 0.71 100 7b >10,000 5 ± 2% at 10 μMn.d. >10,000 15 ± 5% at 10 μM 7c 218 ± 18 86 ± 2  940 ± 114 55 ± 5 0.70± 0.16 45.3 ± 6.8 8c 31.2 ± 3.3 97 ± 1 145 ± 35 96 ± 2 n.d. 9c 142 ± 2491 ± 1 684 ± 75 48 ± 3 0.67 ± 0.19 22.0 ± 2.8^(a)EC₅₀ for stimulation of basal [³⁵S]GTP-γ-S binding by agonists inmembranes from transfected CHO cells (±S.E.M.), n = 3.n.d. not determined.Discussion

Nearly all of the thousands of known adenosine agonists are derivativesof adenosine. Although molecular modeling of adenosine agonists has beencarried out, there has been no direct evidence from this for aconformational preference of the ribose ring in the receptor bindingsite. In the present study, methanocarba-adenosine analogues havedefined the role of sugar puckering in stabilizing the activereceptor-bound conformation. The S-methanocarba analogue of adenosine,5d, was only weakly active, presumably because of a disfavoredconformation that decreases receptor binding. In contrast, themethanocarba analogues constrained in the N-conformation, e.g. 5c-9c,displayed high receptor affinity, particularly at the A3 receptor. Inbinding assays at A1, A2A, and A3 receptors, N-methanocarba-adenosineproved to be of higher affinity than the S-analogue, with anN:S-affinity ratio of 150 at the human A3 receptor. Thus, the biologicalpotency and efficacy of this series of nucleosides appears to be highlydependent on ring puckering, which in turn would influence theorientation of the hydroxyl groups within the receptor binding site.

The structure activity relationship (SAR) of adenosine agonistsindicates that the ribose ring oxygen may be substituted with carbon, asin 5b and 7b, however much affinity is lost. As demonstrated with thearisteromycin derivative, 7b, simple carbocyclic substitution of theribose moiety of otherwise potent, N6-subsituted adenosine agonistsgreatly diminishes affinity, even in comparison to aristeromycin, 5b.

In comparison to the ribose analogues, the N-methanocarba N6-subsitutedadenosine agonists were of comparable affinity at A3 receptors, but lesspotent at A1, A2A, and A2B receptors. The N-methanocarba N6-cyclopentylderivatives were A1 receptor-selective and maintained high efficacy athuman recombinant but not rat brain A1 receptors, as indicated bystimulation of binding of [³⁵S]GTPγS. This may be related to eitherspecies differences or heterogeneity of G proteins, since the degree ofagonist efficacy of a given compound may be highly dependent on thereceptor-associated G protein. N-Methanocarba N6-(3-iodobenzyl)adenosineand the 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3receptors, respectively, and were selective partial agonists. As for theribose parents, additional 2-chloro substitution was favorable forreceptor selectivity. However, unlike the ribose forms, efficacy wasreduced in N6-(3-iodobenzyl) analogues, such that partial A3 receptoragonists 7c and 9c were produced.

Partial agonists are possibly more desirable than full agonists astherapeutic agents due to potentially reduced side effects in theformer. Partial agonists may display in vivo specificity for sites atwhich spare receptors are present, and the drug would therefore behavewith apparent “full” efficacy. Thus, for compounds 7c and 9c, partialagonism combined with unprecedented functional potency at A3 receptors(<1 nM) may give rise to tissue selectivity.

Thus, at least three of the four adenosine receptors favor theN-conformation. For another member of the GPCR superfamily, the P2Y1receptor, we recently reported that the ribose N-conformation of adeninenucleotides also appears to be preferred at the receptor binding site.Thus, the P1 and at least one of the P2 purinoceptors share thepreference for the N-conformation. This may suggest a common motif ofbinding of nucleoside moieties among these GPCRS. The insights of thisconformational preference may be utilized in simulated docking ofadenosine agonists in a putative receptor binding site and to designeven more potent and selective agents.

At the binding site of ADA, the N-isomer is also preferred, although thecarbocyclic adenosine analogues are relatively poor substrates (relativerates of deamination are: 5a, 100; 5b, 0.99; 5c, 0.58; 5d, 0.010.N6-substituted analogues, such as 6c-9c, would not be expected to besubstrates for ADA. Other enzymes, such as HIV reverse transcriptase andHerpes thymidine kinase (HSV-1 TK) are also able to discriminate betweenthe two antipodal conformations of restricted methanocarba thymidineanalogues.

In conclusion, we have found that the introduction of amethano-carbocyclic modification of the ribose ring of purine agonistsrepresents a general approach for the enhancement of pharmacodynamic andbecause of the absence of the glycosyl bond, potentially ofpharmacokinetic properties. This approach could therefore be applied tothe development of cardioprotective, cerebroprotective, andanti-inflammatory agents.

EXAMPLE 2

Introduction

P2 receptors, which are activated by purine and/or pyrimidinenucleotides, consist of two families: G protein-coupled receptors termedP2Y, of which 5 mammalian subtypes have been cloned, and ligand-gatedcation channels termed P2X, of which 7 mammalian subtypes have beencloned. The P2Y₁ receptor, which is present in the heart, skeletal andvarious smooth muscles, prostate, ovary, and brain, was the first P2subtype to be cloned. The nomenclature of P2 receptors and their variousligand specificities is well established.

Nucleotide agonists binding at P2Y₁ receptors induce activation ofphospholipase C (PLC), which generates inositol phosphates anddiacylglycerol from phosphatidyl inositol-(4,5)-bisphosphate, leading toa rise in intracellular calcium. A P2Y₁ receptor antagonist may havepotential as an anti-thrombotic agent, while a selective P2Y₁ receptoragonist may have potential as an anti-hypertensive or anti-diabeticagent.

Recently, progress in the synthesis of selective P2 receptor antagonistshas occurred. Adenosine 3′,5′- and 2′,5′-bisphosphates were recentlyshown to be selective antagonists or partial agonists at P2Y₁ receptors,and other classes of P2 antagonists include pyridoxal phosphatederivatives, isoquinolines, large aromatic sulfonates related to thetrypanocidal drug suramin and various dyestuffs, and2′,3′-nitrophenylnucleotide derivatives. Synthesis of analogues ofadenosine bisphosphates has resulted inN6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (1a, MRS 2179), acompetitive antagonist at human and turkey P2Y₁ receptors, with a KBvalue of approximately 100 Nm. The presence of an N⁶-methyl group andthe absence of a 2′-hydroxyl group both enhanced affinity and decreasedagonist efficacy, thus resulting in a pure antagonist at both turkey andhuman P2Y₁ receptor. The corresponding 2-C1 analogue (1b, MRS 2216) wasslightly more potent than 1a as an antagonist at turkey P2Y₁ receptors,with an IC₅₀ value of 0.22 μM in blocking the effects of 10 nm2-methylthioadenosine-5′diphosphate (2-MeSADP). MRS2179 (compound 1a)was inactive at P2Y₂, P2Y₄, and P2Y₆ subtypes, at the adenylylcyclase-linked P2Y receptor in C6 glioma cells and at a novel avian P2Yreceptor that inhibits adenylyl cyclase. However, the selectivity ofthis series of nucleotides for the P2Y₁ receptor is not absolute, since1a also displayed considerable activity at P2X₁ receptors (EC₅₀ 1.2 μM),but not at P2Y₂₋₄ receptors.

In order to move away from the nucleotide structure of 1a and therebyincrease biological stability and selectivity for the receptors in thepresent study, further structural modifications of the ribose moietyhave been carried out. We have explored the SAR of these two series andintroduced major modifications of the ribose moiety. These modificationsinclude fixing the ring pucker conformation in the carbocyclic seriesusing a bridging cyclopropane ring, ring enlargement with introductionof a nitrogen atom, and ring contraction.

Results

Chemical synthesis

The methanocarbocyclic 2′-deoxyadenosine analogues in which the fusedcyclopropane ring fixes the conformation of the carbocyclic nucleosideinto a rigid northern or southern envelope conformation, as defined inthe pseudoroational cycle, were synthesized as precursors of nucleotides4 and 5 by the general approach of Marquez and coworkers. Again, theN⁶-methyl group was introduced by the Dimroth rearrangement, as shownbelow.

Position adenine modifications were further introduced in theN-configuration series as shown below.

Biological Activity

Adenine nucleotides markedly stimulate inositol lipid hydrolysis byphospholipase C in turkey erythrocyte membranes, through activation of aP2Y₁ receptor. The agonist used in screening these analogues, 2-MeSADP,has a higher potency than the corresponding triphosphate for stimulationof inositol phosphate accumulation in membranes isolated from[³H]inositol-labeled turkey erythrocytes.

The deoxyadenosine bisphosphate nucleotide analogues prepared in thepresent study were tested separately for agonist and antagonist activityin the PLC assay at the P2Y₁ receptor in turkey erythrocyte membranes,and the results are reported in Table 3. Concentration-response curveswere determined for each compound alone and in combination with 10 nM2-MeSADP.

Marquez and coworkers have introduced the concept of ring-constrainedcarbocyclic nucleoside analogues, based on cyclopentane ringsconstrained in the N-(Northern) and S-(Southern) conformations by fusionwith a cyclopropane (methanocarba) ring. In the presnet studies theseries of ring-constrained N-methanocarba derivatives, the 6-NH₂analogues, 4a was a pure agonist of EC₅₀152 nM and 88-fold more potentthan the corresponding S-isomer, 5, also an agonist. Thus, the ribosering N-conformation appeared to be favored in recognition at P2Y₁receptors. The N⁶-methy- and 2-chloro-N⁶-methyl-N-methanocarbaanalogues, 4b and 4c, were antagonists having IC₅₀ values of 276 and 53nM, respectively.

Molecular Modeling

To better understand the role of the sugar puckering on the human P2Y₁agonist and antagonists activities, we carried out a molecular modelingstudy of this new generation of ribose-modified ligands. Suchmodifications include cyclopentyl rings constrained in the N- andS-conformations with cyclopropyl (methanocarba) groups, six-memberedrings (morpholino and anhydrohexitol analogues), and cyclobutylnucleotides. We have recently developed a model of the human P2Y₁receptor, using rhodopsin as a template, by adapting a facile method tosimulate the reorganization of the native receptor structure induced bythe ligand coordination (cross-docking procedure). Details of the modelbuilding are given in the Experimental Section. We have also reportedthe hypothetical molecular basis for recognition by human P2Y₁ receptorsof the natural ligand ATP and the new potent, competitive antagonist2′-deoxy-N⁶-methyladenosine-3′,5′-bisphosphate. Both ATP and 1a arepresent in the hypothetical binding site with a N-sugar ringconformation. In the present work, the sterically constrained N- andS-methanocarba agonist analogues, 4a and 5, respectively, were dockedinto the putative binding site of our previously reported P2Y₁ receptormodel. According to their structural similarity, the cross-dockingprocedure demonstrated that the receptor architecture found for bindingthe ATP and 1a was energetically appropriate also for the binding ofboth 4a and 5. However, N-methanocarba/P2Y₁ complex appeared more stableby approximately 20 kcal/mol than S-methanocarba/P2Y₁ complex. In thelowest energy docked complex of N-methanocarba agonist in the proposedligand binding cavity the side chain of Gln307 is within hydrogenbonding distance of the N⁶ atom at 1.8 Å, and the side chain of Ser314is positioned at 2.0 Å from the N¹ atom and at 3.4 Å from the N⁶ of thepurine ring. As already reported, another three amino acids areimportant for the coordination of the phosphate groups in theantagonist: Arg128, Lys280 and Arg310. Lys280 may interact directly withboth 3′-5′-phosphates (1.7 Å, O3′ and 1.7 Å, O5′), whereas Arg128 andArg310 are within ionic coupling range to both the O₂ and 03 atoms ofthe 5′-phosphate. In molecular modeling studies poor superimposition(rms=1.447) between the N- and S-methanocarba agonist analogues has beenfound inside the receptor binding domain. In particular, the adeninemoiety and 5′ phosphate of the S-methanocarba derivative are shifted outposition relative to with the N-methanocarba isomer, decreasing thestability of the S-methanocarba/PSY₁ complex. This fact might becorrelated with the difference of their biological activity as seen inTable 4 below.

Using the information that a common binding site could be hypothesizedamong these deoxyadenosine bisphosphate analogues, a superimpositionanalysis of the energy-minimized of the more potent antagonists has beenperformed. In this analysis we have used 1a as a reference compound, andwe have defined three matching pairs of atoms, corresponding to N¹ atomof the purine ring and the P atoms of both 3′ and 5′ phosphate groups,to carry out the superimposition analysis. As reported in Table 4,acceptable RMS values have been obtained for all the antagonistscompared with the ^(1a) structure. As shown in FIG. 4A, thissuperimposition study suggested that the two phosphate groups may occupya common receptor regions, and a general pharmacophore model forbisphosphate antagonists binding to the human PSY₁ receptor can beextrapolated.

Discussion

In conclusion the present study has identified new pharmacologicalprobes of PSY₁ receptors, including full agonists, partial agonists, andantagonists. The SAR of 1a indicates that the ribose ring oxygen may bereadily substituted with carbon. Furthermore, analogues of constrainedconformation, e.g. the methanocarba analogues, display enhanced receptoraffinity. Additional 2-chloro and N⁶-methyl substitution is favorablefor affinity at PSY₁ receptors, and nearly pure antagonism is maintainedprovided that the N⁶-methyl group is present.

Thus, the biological potency and efficacy of this series ofbisphosphates appears to be highly dependent on subtle conformationalfactors, which would influence the orientation of the phosphate groupswithin the receptor binding site.

The sugar moiety of nucleosides and nucleotides in solution is known toexist in a rapid, dynamic equilibrium between extreme 2-exo/3′-endo (N-)and 2′-endo/3′-exo (S-) conformations as defined in the pseudorotationalcycle. While the energy gap between N- and S-conformation is in theneighborhood of 4 kcal/mol, such a disparity can explain the differencebetween micromolar and nanomolar binding affinities. Using a molecularmodeling approach, we have analyzed the sugar conformationalrequirements for a new class of bisphosphate ligands binding to thehuman PSY₁ receptor. As experimentally shown, the ribose ring Northernconformation appeared to be favored in recognition at human PSY₁receptor (see Table 4). We have found new support to our recentlypresented hypothesis in which three important recognition regions arepresent in the bisphosphate molecular structures; The N¹ atom of thepurine ring and the P atoms of both 3′ and 5′ phosphate groups. TheN-conformation seems to be essential to maximize the electrostaticinteractions between the negatively charged phosphates and thepositively charged amino acids present in the receptor binding cleft, aswell Arg128, Lys280, and Arg310.

Interestingly, the electrostatic contacts also appear to be crucial forthe recognition of bisphosphate antagonists. Using superimpositionanalysis, a general pharmacophore model for the bisphosphate antagonistsbinding to the PSY₁ receptor has been proposed. According to thepharmacophore map, recognition of the bisphosphates antagonists at acommon region inside the receptor binding site and, consequently, acommon electrostatic potential profile is possible. As well for theagonists, the Northern conformation seems to be essential to maximizethe electrostatic interactions between the negatively charged phosphatesand the positively charged amino acids presents in the receptor bindingcleft. As we predicted using the previously reported PSY₁ receptormodel, sugar moiety does not seen to be crucial for the ligandrecognition process.

As already described, the simple addition of the N⁶-methyl group inseveral cases converted pure agonists to antagonists. From apharmacological point of view, this is really a unique situation. Withthe addition of the N⁶-methyl group it is not possible to have a doublehydrogen-bonding interaction and, consequently, the activation pathwayis blocked. However, for all the N⁶-methyl antagonists the possibilityto participate in at least one of the two possible hydrogen bondsappears to be very important for the increase in affinity at the PSY₁receptor.

Chemical Synthesis

Nucleosides and synthetic reagents were purchased from Sigma ChemicalCo. (St. Louis, Mo.) and Aldrich (St. Louis, Mo.).6-Chloro-2′-deoxypurine riboside was obtained from Sigma. Several2′-deoxynucleosides, including an anhydrohexitol-adenine nucleoside and2′-deoxyaristeromycin were also synthesized.

Purity of compounds was checked using a Hewlett-Packard 1090 HPLCapparatus equipped with an SMT OD-5-60 RP-C18 analytical column (250×4.6mm; Separation Methods Technologies, Inc., Newark, Del.) in two solventsystems. System A: Linear gradient solvent system: 0.1 M TEAA/CH₃CN from95/5 to 40/60 in 20 min and the flow rate was of 1 mL/min. System B:linear gradient solvent system: 5 mM TBAP/CH₃CN from 80/20 to 40/60 in20 min and the flow rate was of 1 mL/min. Peaks were detected by UVabsorption using a diode array detector. All derivatives showed morethan 95% purity in the HPLC systems.

Purification of most of the nucleotide analogues for biological testingwas carried out on DEAE-A25 Sephadex columns as described above.However, compounds 7b and 8a-c required HPLC purification (system a,semi-preparative C18 column) of the reaction mixtures.

General Procedure of Phosphorylation.

Method A: The nucleoside (0.1 mmol) and Proton Sponge® (107 mg, 0.5mmol) were dried for several h in high vacuum at room temperature andthen suspended in 2 mL of trimethyl phosphate. Phosphorous oxychloride(Aldrich, 37 μL, 0.4 mmol) was added, and the mixture was stirred for 1h at 0° C. The reaction was monitored by analytical HPLC (eluting with agradient consisting of buffer: CH₃CN in the ratio 95:5 to 40:60, inwhich the buffer was 0.1 M triethylammonium acetate (TEAA); elution timewas 20 min; flow rate was 1 mL/min; column was SMT OD-5-60 RP-C18;detector was by UV in the E_(max) range of 260-300 nm). The reaction wasquenched by adding 2 mL of triethylammonium bicarbonate buffer and 3 mLof water. The mixture was subsequently frozen and lyophilized.Purification was performed on an ion-exchange column packed withSephadex-DEAE A-25 resin, a linear gradient (0.01 to 0.5 M) of 0.5 Mammonium bicarbonate was applied as the mobile phase, and UV and HPLCwere used to monitor the elution. All nucleotide bisphosphates werecollected, frozen and lyophilized as the ammonium salts. All synthesizedcompounds gave correct molecular mass (high resolution FAB) and showedmore than 95% purity (HPLC, retention times are reported in Table 4).

Method B: Nucleoside (0.1 mmol) dried for several h in high vacuum atroom temperature was dissolved in 2 mL of dry THF. Lithiumdiisopropylamide solution (Aldrich, 2.0 M in THF, 0.4 mmol) was addedslowly at −78° C. After 15 min tetrabenzyl pyrophosphate (Aldrich, 0.4mmol) was added and the mixture was stirred for 30-60 min at −78° C. Thereaction mixture was warmed to 0° C.-rt and stirred for an additionperiod ranging from 2 h to 24 h. Chromatographic purification (pTLC,CHCl₃:CH₃OH(10:1) gave the tetrabenzyl phosphorylated compound. Thiscompound (20 mg) was dissolved in a mixture of methanol (2 mL) and water(1 mL) and hydrogenated over a 10% Pd-on-C catalyst (10 mg) at rt for 62h. The catalyst was removed by filtration and the methanol wasevaporated. The residue was treated with ammonium bicarbonate solutionand subsequently frozen and lyophilized. Purification, if necessary, wasby the same procedure as in method A.

(N-Methanocarba-2′-deoxyadenosine-3′,5′-bis(diammonium phosphate) (4a)[(IR,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-aminopurin-9-yl)bicyclo[3.1.0.]-hexane-2-phosphatetetraammonium salt]

Starting from 16 mg (0.06 mmol) of (N)-methanocarba-2′deoxyadenosine andfollowing the general phosphorylation procedure A we obtained 1.8 mg(0.0037 mmol, 5.5% yield) of the desired compound.

¹H-NMR (D₂O) ∂ 0.90 (1H, m, CH₂-6), 1.10 (1H, m CH₂6′), 1.82 (1H, m,CH-5), 1.91 (1H, m, CH₂-3′) 2.23 (1H, m, CH₂-3′), 3.49 (1H, d, J=11.7Hz, CH₂—OH), 4.16 (1H, d, J=6.9 Hz, CH₂-2′), 8.39 (1H, s, H-2), 8.54(1H, s, H-8).

³¹P-NMR (D₂O) ∂ 0.43 (s, 5′P); −0.19 (s, 3′P).

(N)-Methanocarba-N⁶-methyl-2′deoxyadenosine-3′,5′-bis(diammoniumphosphate) (4b)(1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(6-methylaminopurin-9-yl)bicyclo[[3.1.0.]-hexane-2-phosphatetetraammoniun salt]

13.5 mg (0.0170 mmol) of compound 18 was converted to the correspondingphosphoric acid analog using hydrogenation following the generalprocedure B. Purification was performed on an ion-exchange column packedwith Sephadex-DEAF A-25 resin, linear gradient (0.01 to 0.5 M) of 0.5 Mammonium bicarbonate was applied as the elan to give 3.0 mg (0.0060mmol, 35.3% yield) of the desired compound.

¹H-NMR (D₂P) ∂ 0.93-0.98 (1H, m, CH₂-6′), 1.17 (1H, m, CH₂-6′),1.86-1.88 (1H, m, CH5′), 1.94-1.98 (1H, m, CH₂-3′), 2.23-2.31 (1H, m,CH₂-3′), 3.09 (3H, bs, N⁶—CH₃), 3.61-3.64 (1H, m, CH₂OH), 4.51-4.55 (1H,m, CH₂OH), 5.01-5.03 (1H, m, CH-4′), 5.19-5.21 (1H, m, CH-2′), 8.22 (1H,s, H-2), 8.51 (1H, s, H-8). 31P-NMR (D₂O) ∂ 1.26, 1.92 (2s, 3′-P, 5′-P).

(N)-Methanocarba-N⁶-methyl-2-chloro-2′-deoxyadenosine-3′,5′-bis(diammoniumphosphate) (4c)[(1R,2S,4S,5S)-1[(phosphato)methyl]-4-(2-chloro-6-aminopurin-9-yl)bicyclo[3.1.0]-hexane-2-phosphate tetraammonium salt]

The nucleoside, compound 23, reacted with tetrabenzyl pyrophosphate, asin Method B, followed by an alternative deprotection procedure. Startingfrom 10 mg (0.0323 mmol) of(N)-methanorcarba-N⁶-methyl-2-chloro-2′-deoxyadenosine and following thegeneral phosphorylation procedure (Method B) we obtained 9.5 mg (0.0114mmol, 35.3% yield) of the desired compound,(N)-methanocarba-N⁶-methyl-2-chloro-2′-deoxyadenosine-3′,5′-bis(dibenzylphosphate).

¹H-NMR (CDCI₃)∂ 0.75-0.81 (H, m,CH₂-6′), 103-1.08 (1H, m, CH₂-6′),1.49-1.51 (1H, m, CH-5′), 1.84-1.94 (1H,m, CH₂-3′), 1.99-2.10 (1H, m,CH₂-3′), 3.12 (3H, bs, N⁶—CH₃), 4.11-4.20 (1H, m, CH₂OH), 4.50-4.55 (H,m, CH₂OH), 4.90-4.98, (8H, m, —OCH₂), 4.99-5.01 (1H, m, CH-4′),5.23-5.30 (1H, m, CH-2′), 5.90 (1H, BS, NH), 7.20-7.29 (20H, m, C₆H₅),7.82 (1H, s, H-8)

³¹P-NMR (D₂O) ∂ −0.58 (s,5′P); −1.06 (s,3′P).

MS(CI—NH₃) (M+1) 830 HRMS (FAB−) (M+Cs) Calcd. 962.1252; Found 962, 1252

9.5 mg (0.0114 mmol) of the tetrabenzyl-protected intermediate added todry CH₂Cl₂ (1.0 mL) was cooled to −78° C. under argon and treated with100 μL of boron trichloride solution (1M in CH₂Cl₂) and 100 μL ofanisole. The reaction mixture was stirred for 12 hr at 0° C. to rt andextracted with triethylamine solution. Purification was performed on anion-exchange column packed with Sephadex-DEAE A-25 resin, lineargradient (0.01 to 0.5 M) of 0.5 M ammonium bicarbonate was applied asthe eluent to give 0.4 mg (0.0007 mmol, 6.52 yield) of the desiredcompound 4c.

¹H-NMR (D₂O) ∂0.91-0.96 (1H, m, CH₂-6′), 1.12-1.16 (1H, m, CH₂-6′),1.80-1.84 (1H, m, CH-5′), 1.85-1.98 (1H, m, CH₂-3′), 2.20-2.50 (1H, m,CH₂-3′), 3.08 (3H, bs, N⁶—CH₃), 3-57-3.60 (1H, m, CH₂OH), 4.52-4.67 (1H,m, CH₂OH), 4.94-4.96 (1H, m, CH-4′), 5.18-5.21 (1H, m, CH-2′), 8.52 (1H,s,H-8)

³¹P-NMR (D₂O) ∂ 1.82, 2.52 (2s, 3′-P, 5′P)

(S)-Methanocarba-2′, deoxyadeonosine-3′,5′-bis(diammonium phosphate (5)[(1S,3S,4R,5S)-4-[(phosphato)methyl]-1-(6-aminopurin-9-yl)bicyclo[3.1.0]-hexane-3-phosphatetetraammonium salt]

Starting from 16 mg (0.06 mmol) of (S)-methanocarba-2′deoxyadenosine andfollowing the general phosphorylation procedure A, we obtained 2.1 mg(0.0043 mmol, 7.55 yield) of the desired compound 5.

¹H-NMR (D₂O) ∂ 1.36 (1H, m, CH₂-6′), 1.53 (1H, t, J=4.8 Hz, CH₂-6′),2.05 (1H, m, CH₂-5′), 2.30 (1H, m, CH-4′), 2.46 (2H, m, CH₂-2′), 3.97(2H, m, CH₂OH), 4.45 (1H, d, J=6.6 Hz, CH-3′), 8.16 (1H, s, H-2), 8.30(1H, s, H-8). ³¹P-NMR (D₂O) ∂ 0.85 (bs, 5′P); 0.31 (bs, 3′P).

[(1S,3S,4R,5S)-1-[(Hydroxy)methyl]-2-hydroxy-4-(6-methylaminopurin-9-1yl)bicyclo[3.1.0]-hexane(17b)

The Dimroth rearrangement (Scheme 2) was carried out on(N)-methanocarba-2′-deoxyadenosine. Specifically, the(N)-methanorcarba-2′-deoxyadenosine (17a, 50.0 mg, 0.191 mmol) washeated at 40° C. with methyl iodide (71.5 μL, 1.15 mmol) in dry DMF (2.0mL) for 48 h. The solvent was evaporated under reduced pressure, and theresidue was heated at 90° C. with ammonium hydroxide (4.0 mL) for 4 h.The water was evaporated, and the residue was purified by pTLC usingMeOH; CHCl₃ (1:9) to afford compound 17b as a colorless solid (40 mg,0.15 mmol, 76%).

¹H-NMR (CD₃OD) ∂ 0.77.-0.81 (1H, m, CH₂-6′), 1.03-1.07 (1H, m, CH₂-6′),1.68-1.72 (1H, m, CH-5′), 1.79-1.89 (1H, m, CH₂-3′), 2.00-2.07 (1H, m,CH₂-3′), 3.12 (3H, bs, N⁶—CH₃), 3-33 (1H, d, J=CH₂OH), 4.29 (1H, d,J=11.7 Hz, CH₂OH), 4.89-4.92 (1H, m, CH-4′), 5.02 (1H, d, J=6.9 Hz,CH-2′), 8.24 (1H, s,H-2), 8.49 (1H, s, H-8).

MS(CI—NH₃): 276 (M+1) 830 HRMS(FAB−) (M+Cs) Calcd. 275.1382; Found275.1389.

(N)-Methanocarba-N⁶-methyl-2′-deoxyadenosine-3′,5′-bis(dibenzylphosphate)(18)[(1S,2S,4S,5S)-1-[(dibenzylphosphato)methyl]-4-(6-methylaminopurin-9-yl)bicyclo[3.1.0]-hexane-2-dibenzylphosphate]

Starting from 20.0 mg (0.0726 mmol) ofN-methanorcarba-N⁶-methyl-2′-deoxyadenosine 17b and following thegeneral phosphorylation procedure (Method B we obtained 13.5 mg (0.0170mmol, 23.4% yield) of the desired protected intermediate, 18 as shown inScheme 2.

¹H-NMR (CDCI₃) ∂ 0.73-0.78 (1H, m, CH₂-6′), 0.94-0.98 (1H, m, CH₂-6′),1.53-1.54 (1H, m, CH-5′), 1.81-1.91 (1H, m, CH₂-3′), 2.05-2.13 (1H, m,CH₂-3′), 3.15 (3H, bs, N⁶—CH₃), 3-70-3.83 (1H, m, CH₂OP), 4.49-4.55 (1H,m, CH₂OP), 4.89-5.00 (8H, m, OCH-₂), 5.02-5.06 (1H, m, CH-4′), 5.27-5.32(1H, m, CH-2′), 5.86 (1H, bs, NH), 7.21-7.23 (20H, m, C₆H₅), 7.86 (1H,s,H-2), 8.31 (1H, s,H-8). ³¹P-NMR (D₂O) ∂ −0.56, −1.05 (2s, 3′-P, 5′P)HRMS (FAB−) (M−Cs) Calcd. 928.1641; Found 928.1700.

[(1S,2S,42,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-6-dichloropurin-9-yl)bicyclo[3.1.0]-hexane(21)

To an ice cold solution of triphenylphosphine (278 mg, 1.06 mmol) in dryTHF (2 mL) was added diethylazadicarboxylate (170, 1.06 mmol) dropwiseunder a nitrogen atmosphere, and the mixture was stirred for 20 minuntil the solution turned red orange (Scheme 3). This mixture was addeddropwise to a cold stirred mixture of the starting alcohol (135 mg,0.417 mmol) and 2.6-dichloropurine (157 mg, 0.883 mmol) under a nitrogenatmosphere. The reaction mixture was stirred in an ice bath for 30 minand then allowed to warm to room temperature, and stirring continued for12 h. Solvent was removed by nitrogen purge, and the residue waspurified by pTLC using EtOAc petroleum ether (1:1) to afford a thickliquid (132 mg, 0.263 mmol, 64%).

¹H NMR: (CD₃OD) δ 0.85 (m, 1H), 1.13 (m, 1H), 1.59 (m, 1H), 1.68 (m,1H), 2.06 (m, 1H), 3.17 (d, J=10.8 Hz, 1H), 4.11-4.57 (m, 5H), 5.20 (d,J=6.9 Hz, 1H), 6.6 (bs, 1H), 7.23-7.37 (m, 10H), 8.98 (s, 1H).

MS: (EI) 494 (M+).

[(1R,2S,4S,5S)-1-[(Benzyloxy)methyl]-2-benzyloxy-4-(2-chloro-6-methylaminopurin-9-yl)bicyclo[3.1.0]-hexane(22)

Compound 21 (100 mg, 0.202 mmol) was dissolved in methylamine inmethanol (30% solution, 3 mL) and was stirred at rt for 12 h under anitrogen atmosphere. The solvent was evaporated, and the crude productwas purified by pTLC using EtOAc:petroleum ether (6:4) to afford 22 as alight yellow solid (86 mg, 0.176 mmol, 88%).

¹H NMR: (CD₃OD) δ 0.70 (m, 1H), 1.06 (m, 1H), 1.50 (m, 1H), 1.76 (m,1H), 1.96 (m, 1H), 3.01 (s, 3H), 3.08 (m, 2H), 4.03 (m, 4H), 4.45 (bs,1H), 5.02 (bs, 1H), 8.38 (s, 1H).

MS: (Cl): 490 (M+1).

[(1R,2S,42,5S)-1-[(Hydroxy)methyl]-2-hydroxy-4-(2-chloro-6-methylaminopurin-9-yl)bicyclo[3.1.0]-hexane(23)

Compound 22 (40 mg 0.0816 mmol) was dissolved in dry CH₂Cl₂ (1.0 mL),and hydrogenated using BCl₃ (1M in CH₂Cl₂, 175 μL) for 50 min at −78° C.under argon. The solvent was evaporated, and the crude product waspurified by pTLC using CHCl₃: MeOH (10:1) to afford 23 as a light yellowsolid (10.0 mg, 0.0323 mmol, 39.6%).

¹H NMR: (CD₃OD) ∂ 0.77-0.81 (1H, m, CH₂-6′), 1.02-1.05 (1H, m, CH₂-6′),1.65-1.68 (1H, m, CH-5′), 1.78-1.91 (1H, m, CH₂-3′), 1.99-2.07 (1H, m,CH₂-3′), 3.08 (3H, bs, N⁶—CH₃), 3.37 (1H, d, J=11.7 Hz, CH₂OH), 4.27(1H, d, J=11.7 Hz, CH₂OH), 4.89-4.91 (1H, m, CH-4), 4.97 (1H, d, J=6.8Hz, CH-2′), 8.46 (1H, s, H-8).

MS: (CI—NH₃): 310 (M+1), HRMS (FAB−): Calcd 309.0992, Found 309.0991.

Pharmacological Analyses.

P2Y₁ receptor promoted stimulation of inositol phosphate formation byadenine nucleotide analogues was measured in turkey erythrocytemembranes as previously described. The K_(0.5) values were averaged from3-8 independently determined concentration-effect curves for eachcompound. Briefly, 1 mL of washed turkey erythrocytes was incubated ininositol-free medium (DMEM; Gibco, Gaithersburg Md.) with 0.5 mCi of2-[³H]myo-inositol (20Ci/mmol: American Radiolabelled Chemicals, Inc.,St. Louis Mo.) for 18-24 h in a humidified atmosphere of 95% air/5% CO₂at 37° C. Erythrocyte ghosts were prepared by rapid lysis in hypotonicbuffer (5 mM sodium phosphate, pH 7.4, 5 mM MgCl₂, 1 mM EGTA) asdescribed. Phospholipase C activity was measured in 25 μL of[³H]inositol-labeled ghosts (approximately 175 μg of protein,200-500,000 cpm/assay) in a medium containing 424 μM CaCl₂, 0.91 mMMgSO₄, 2 mM EGTA, 115 mM KCl, 5 mM KH₂PO₄, and 10 mM Hepes pH 7.0.Assays (200 μL final volume) contained 1 μM GTPγS and the indicatedconcentrations of nucleotide analogues. Ghosts were incubated at 30° C.for 5 min, and total [³H]inositol phosphates were quantitated by anionexchange chromatography as previously described.^(7,36)

Data Analysis.

Agonist potencies were calculated using a four-parameter logisticequation and the GraphPad software package (GraphPad, San Diego,Calif.). EC₅₀ values (mean±standard error) represent the concentrationat which 50% of the maximal effect is achieved. Relative efficacy (%)was determined by comparison with the effect produced by a maximaleffective concentration of 2-MeSADP in the same experiment.

Antagonist IC₅₀ values (mean±standard error) represent the concentrationneeded to inhibit by 50% the effect elicited by 10 nM 2-MeSADP. Thepercent of maximal inhibition is equal to 100 minus the residualfraction of stimulation at the highest antagonist concentration.

All concentration-effect curves were repeated in at least three separateexperiments carried out with different membrane preparations usingduplicate or triplicate assays. TABLE 3 Stimulation of PLC at turkeyerythrocyte P2Y₁ receptors (agonist effect) and the inhibition of PLCstimulation elicited by 10 nM 2-MeSADP (antagonist effect), for at leasttwo separate determinations. Agonist Antagonist Effect, Effect, % of %of maximal maximal IC₅₀, μM^(b) Compound increase^(a) EC₅₀, μM^(a)inhibition^(b) (n) 1a^(c,e) NE 99 ± 1 0.331 ± 0.059 (MRS 2179) (5)1b^(e) NE 95 ± 1 0.206 ± 0.053 1c^(e) 4 d 96 ± 2 1.85 ± 0.74 1d^(e) 6 ±2 d 94 ± 2 0.362 ± 0.119 4a 92 ± 5  0.155 ± 0.021 NE 4b NE 100 0.157 ±0.060 4c NE 100 0.0516 ± 0.0008 5 41 ± 13 13.3 34% at small decrease 100μM^(a)Agonist potencies were calculated using a four-parameter logisticequation and the GraphPad softaware package (GraphPad, San Diego, CA).EC₅₀ values (mean ± standard error) represent the concentration at which50% of the maximal effect is achieved. Relative efficacies (%) weredetermined by comparison with the effect produced by a maximal effectiveconcentration of 2-MeSADP in the same experiment. Small increase refersto <10% at 100 μM.^(b)Antagonist IC₅₀ values (mean ± standard error) represent theconcentration needed to inhibit by 50% the effect elicited by 10 nM2-MeSADP. The percent of maximal inhibition is equal to 100 minus theresidual fraction of stimulation at the highest antagonistconcentration.^(c)1a, MRS 2179; 4c, MRS 2279.d EC₅₀ was not calculated for increases of ≦10% at 100 μM.^(e)values from refs. 17, 19.NE no effect at 100 μM.

TABLE 4 Synthetic data for nucleotide derivatives, including structuralverification using high resolution mass spectroscopy and purityverification using HPLC. FAB (M − H⁺) HPLC (rt; min)^(a) No FormulaCalcd Found System A System B Method, Yield (%)^(b) 2 C₁₀H₁₅O₉N₅P₂410.0267 410.0269 3.53 10.72 B, 21.7 3b C₁₂H₁₉O₈N₅P₂ 422.0631 422.06643.41 8.21 B, 8.0 4a C₁₂H₁₇O₈N₅P₂ 420.0474 420.0482 3.92 7.30 A, 5.5 4bC₁₃H₁₉O₈N₅P₂ 434.0631 434.0622 5.91 7.83 B, 8.3 4c C₁₃H₁₈O₈N₅P₂Cl468.0241 468.0239 8.05 8.54 B, 2.3 5 C₁₂H₁₇O₈N₅P₂ 420.0474 420.0481 4.026.84 A, 7.5 6 C₁₁H₁₆O₈N₅P₂Cl 442.0084 442.0070 6.67 6.82 A, 24.3 7bC₁₂H₂₀O₁₂N₅P₃ 518.0237 518.0243 4.98 12.74 A, 1.8 7c C₁₂H₁₉O₉N₅P₂438.0580 438.0580 4.63 9.36 B, 50.1 7d C₁₂H₁₈O₉N₅P₂Cl 472.0201 472.01905.67 9.97 B, 31.3 8a C₁₂H₂₀O₈N₆P₂ 437.0740 437.0721 2.37 8.78 8.0 8bC₁₂H₂₁O₁₁N₆P₃ 517.0403 517.0404 2.,42 9.23 7.2 8c C₁₂H₂₂O₁₄N₆P₄ 597.0066597.0053 2.96 10.02 4.0^(a)Purity of each derivative was ≧95%, as determined using HPLC withtwo different mobile phases. System A: gradient of 0.1M TEAA/CH₃CN from95/5 to 40/60 and System B: gradient of 5 mM TBAP/CH₃CN from 80/20 to40/60.^(b)Phosphorylation methods: Method A refers to use of phosphorousoxychloride, and Method B refers to use of tetrabenzylpyrophosphate/lithium diisopropylamide followed by hydrogenation. Thepercent yields refer to overall yield for each phosphorylation sequence.For the method of synthesis of 8 refer to Experimental Section.AbbreviationsAIBN, 2,2′-azobisisobutyronitrile;ATP, adenosine 5′-triphosphate;DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;DCTIDS, 1,3-dichlorotetraisopropyl-1,1,3,3,-disiloxane;DEAD, diethylazadicarboxylate;DEAE, diethylaminoethyl;DMAP, 4-dimethylaminopyridine;DMF, dimethylformamide;DMSO, dimethylsulfoxide;FAB, fast atom bombardment (mass spectroscopy);HPLC, high pressure liquid chromatography;MS, mass spectroscopy;HRMS, high resolution mass spectroscopy;LDA, lithium diisoproylamide;2-MeSADP, 2-methylthioadenosine-5′-diphosphate;TBAP, tetrabutylammonium phosphate;TEAA, triethylammoniun acetate;THF, tetrahydrofuran;

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1. A compound having the formula A-M, wherein A is a chemically modifiedadenine or uracil and M is a constrained cycloalkyl group, said adenineor uracil is bonded to said constrained cycloalkyl group, and saidcompound binds a receptor; or a salt of said compound.
 2. The compoundof claim 1, wherein said receptor is a P1 or P2 receptor.
 3. Thecompound of claim 2, wherein said P1 receptor is selected from the groupconsisting of A₁, A_(2A), and A₃.
 4. The compound of claim 2, whereinsaid P2 receptor is selected from the group consisting of P2Y and P2X.5. The compound of claim 1, wherein said constrained cycloalkyl groupincludes a cyclopentyl group.
 6. The compound of claim 3, wherein saidconstrained cyclopentyl group is a cyclopentyl ring derivatized with afused cyclopropane bridge.
 7. The compound of claim 1, wherein saidconstrained cycloalkyl group is constrained in the N-conformation. 8.The compound of claim 1, wherein said constrained cycloalkyl group isconstrained in the S-conformation.
 9. A compound of the formula

wherein R₁ is hydrogen, alkyl, cycloalkyl, alkoxy, cycloalkoxy, aryl,arylalkyl, acyl, sulfonyl, arylsulfonyl, thiazolyl or bicyclic alkyl; R₂is hydrogen, halo, alkyl, aryl, arylamino, aryloxide, alkynyl, alkenyl,thiol, cyano, or; R₃, R₄, and R₅, are each independently hydrogen,hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino,arylamino, phosphoryl, diphosphoryl, triphosphoryl, phosphonyl, boronyl,thiophosphoryl, thiodiphosphoryl, thiotriphosphoryl or vanadyl, and canbe the same or different; R₆ is hydrogen, alkyl, alkenyl, alkynyl,heteroaryl or aminoalkyl; R₇ is methylene, dihalomethyl, carbonyl,sulfoxide; and at least one of R₁, R₂, and R₆ is other than hydrogen; R₈is carbon or nitrogen; or a salt of said compound.
 10. The compound ofclaim 9, wherein R₁ is alkyl, cycloalkyl, alkoxy, aryl, arylalkyl,bicycloalkyl, or sulfonyl.
 11. The compound of claim 9, wherein R₁ ismethyl, cyclopentyl, cyclohexyl, phenyl, R-phenylisopropyl, benzyl, orphenylethyl; R₂ is chloro; and R₆ is C₁-C₆ alkylamino, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl.
 12. The compound of claim 9, wherein R₁ isfurther substituted with a member selected from the group consisting ofhydroxyl, halo, sulfonyl, amino, cyano, alkoxyl, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, arylalkyl, sulfonamido, carboxyl, andcarboxamido.
 13. The compound of claim 9, wherein R₁ is methyl group andR₂ is chloro, alkylthio, or arylalkylthio.
 14. The compound of claim 9,wherein R₆ is methyl and R₂ is chloro, alkylthio, arylalkylthio orhydrogen.
 15. The compound of claim 9, wherein R₆ is halo and R₂ is achloro, alkylthio, arylalkylthio or hydrogen.
 16. The compound of claim9, wherein R₂ is chloro.
 17. The compound of claim 9, wherein R₁ ismethyl and R₂ is chloro and R₃ is hydrogen. 18-20. (canceled)
 21. Acompound selected from the group consisting of:

wherein R₁, R₂, R₉ is hydrogen, alkyl, alkenyl, alkynyl, aminoalkyl andR₃, R₄, and R₅, are each independently hydrogen, hydroxyl, alkoxy,alkyl, alkenyl, alkynyl, aryl, acyl, alkylamino, arylamino, phosphoryl,phosphonyl, boronyl, or vanadyl, and can be the same or different; R₆and R₇ are each independently sulfur or oxygen; and R₁₀ is methylene,dihalomethyl, carbonyl, sulfoxide; or a salt of said compound.
 22. Thecompound of claim 21, wherein R₁ is methyl.
 23. A compound comprising amethanocarbocyclic analog of a chemically modified adenosine or uradinewherein said compound is a P2 receptor ligand; or a salt of saidcompound.
 24. The compound of claim 23, wherein the compound is a P2receptor agonist.
 25. The compound of claim 23, wherein the compound isa P2 receptor antagonist.
 26. The compound of claim 22, wherein said P2receptor is selected from the group consisting of P2Y and P2X.
 27. Thecompound of claim 22, wherein said P2 receptor is a P2Y receptor. 28.The compound of claim 22, wherein said P2 receptor is a P2Y1 receptor.29. The compound of claim 22, wherein said P2 receptor is a P2Xreceptor.
 30. A compound comprising a methanocarbocyclic analog of achemically modified adenosine or uradine wherein said compound is a P1receptor ligand; or a salt of said compound.
 31. The compound of claim30, wherein the compound is a P1 receptor agonist.
 32. The compound ofclaim 30, wherein the compound is a P1 receptor antagonist.
 33. Thecompound of claim 30, wherein said P1 receptor is selected from thegroup consisting of A₁, A₂A, and A₃.
 34. The compound of claim 30,wherein said P1 receptor is A₁ receptor.
 35. The compound of claim 30,wherein said P1 receptor is A₃ receptor.
 36. A method of treating orpreventing in a mammal a disease, state, or condition that responds toan adenosine, ATP, or UTP receptor agonist or antagonist comprisingadministering to the mammal a compound of claim
 1. 37. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 1. 38-39. (canceled)
 40. A method for the treatment ofairway diseases, cancer, cardiac arrhythmia, cardiac ischemia, epilepsy,Huntington's Disease, immunodeficient disorders, inflammatory disorders,neonatal hypoxia, neurodegenerative, pain, Parkinson's Disease, renalfailure, schizophrenia, sleep disorders, stroke, thrombosis, urinaryincontinence, diabetes, psoriasis, septic shock, brain trauma, glaucoma,or congestive heart failure in individuals in need of such treatmentcomprising contacting an effective quantity of a compound of claim 1.41. A method of treating or preventing in a mammal a disease, state, orcondition that responds to an adenosine, ATP or UTP receptor agonist orantagonist comprising administering to the mammal a compound of claim 9.42. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a compound of claim
 9. 43. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 21. 44. A method of treating or preventing in a mammala disease, state, or condition that responds to an adenosine, ATP, orUTP receptor agonist or antagonist comprising administering to themammal a compound of claim
 21. 45. A method for the treatment of airwaydiseases, cancer, cardiac arrhythmia, cardiac ischemia, epilepsy,Huntington's Disease, immunodeficient disorders, inflammatory disorders,neonatal hypoxia, neurodegenerative, pain, Parkinson's Disease, renalfailure, schizophrenia, sleep disorders, stroke, thrombosis, urinaryincontinence, diabetes, psoriasis, septic shock, brain trauma, glaucoma,or congestive heart failure in individuals in need of such treatmentcomprising contacting an effective quantity of a compound of claim 9.46. A method for the treatment of airway diseases, cancer, cardiacarrhythmia, cardiac ischemia, epilepsy, Huntington's Disease,immunodeficient disorders, inflammatory disorders, neonatal hypoxia,neurodegenerative, pain, Parkinson's Disease, renal failure,schizophrenia, sleep disorders, stroke, thrombosis, urinaryincontinence, diabetes, psoriasis, septic shock, brain trauma, glaucoma,or congestive heart failure in individuals in need of such treatmentcomprising contacting an effective quantity of a compound of claim 21.